Peter Paulsen

Survival of Campylobacter jejuni under Conditions of Atmospheric Oxygen Tension with the Support of Pseudomonas spp.

ABSTRACT Campylobacter jejuni is a major food-borne pathogen. Despite causing enteritis in humans, it is a well-adapted intestinal microorganism in animals, hardly ever generating disease symptoms. Nevertheless, as a true microaerophilic microorganism it is still puzzling how Campylobacter cells can survive on chicken meat, the main source of human infection. In this study, we demonstrate that C. jejuni is able to withstand conditions of atmospheric oxygen tension when cocultured with Pseudomonas species, major food-spoiling bacteria that are frequently found on chicken meat in rather high numbers. Using an in vitro survival assay, interactions of 145 C. jejuni wild-type strains and field isolates from chicken meat, broiler feces, and human clinical samples with type strains and food isolates of Pseudomonas spp., Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, and Enterococcus faecalis were studied. When inoculated alone or in coculture with Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, or Enterococcus faecalis type strains, Campylobacter cells were able to survive ambient oxygen levels for no more than 18 h. In contrast, Campylobacter bacteria inoculated with type strains or wild-type isolates of Pseudomonas showed a prolonged aerobic survival of up to >48 h. This microbial commensalism was diverse in C. jejuni isolates from different sources; isolates from chicken meat and humans in coculture with Pseudomonasputida were able to use this survival support better than fecal isolates from broilers. Scanning electron microscopy revealed the development of fiberlike structures braiding P. putida and C. jejuni cells. Hence, it seems that microaerophilic C. jejuni is able to survive ambient atmospheric oxygen tension by metabolic commensalism with Pseudomonas spp. This bacterium-bacterium interaction might set the basis for survival of C. jejuni on chicken meat and thus be the prerequisite step in the pathway toward human infection.

Preliminary Analysis of Tetracycline Residues in Marketed Pork in Hanoi, Vietnam

Abstract:  A cross‐sectional survey was designed to investigate the proportion of tetracycline residues in marketed pork in suburb and urban districts in Hanoi. A total of 290 raw muscle samples were randomly collected from open markets in these districts. The samples were qualitatively screened for tetracycline residues using the agar inhibition test, and Bacillus cereus (ATCC 11778) as the reference strain. The inconclusive samples were then analyzed using high‐performance liquid chromatography (HPLC). The positive samples from either test were defined as positive results. Overall, 5.5% of all collected samples were positive for tetracycline residues. The proportion of positive samples from shops in suburb districts was significantly (P < 0.05) different from those collected from shops in urban districts. So, the factor of region was identified as a risk factor of tetracycline residue proportion in raw pork with an odds ratio (OR) of 4.03 (95% CI = 1.12, 14.45). For the other factors, such as season, type of shop, type of abattoir, origin of meat, etc., the difference in proportion of positive samples within each factor was substantial but not statistically significant. These factors were identified as nonrisk factors. Such a high proportion may pose a potential hazard to public health, particularly since they might induce drug resistance of pathogenic micro‐organisms.

Bacteriophages isolated from chicken meat and the horizontal transfer of antimicrobial resistance genes

ABSTRACT Antimicrobial resistance in microbes poses a global and increasing threat to public health. The horizontal transfer of antimicrobial resistance genes was thought to be due largely to conjugative plasmids or transposons, with only a minor part being played by transduction through bacteriophages. However, whole-genome sequencing has recently shown that the latter mechanism could be highly important in the exchange of antimicrobial resistance genes between microorganisms and environments. The transfer of antimicrobial resistance genes by phages could underlie the origin of resistant bacteria found in food. We show that chicken meat carries a number of phages capable of transferring antimicrobial resistance. Of 243 phages randomly isolated from chicken meat, about a quarter (24.7%) were able to transduce resistance to one or more of the five antimicrobials tested into Escherichia coli ATCC 13706 (DSM 12242). Resistance to kanamycin was transduced the most often, followed by that to chloramphenicol, with four phages transducing tetracycline resistance and three transducing ampicillin resistance. Phages able to transduce antimicrobial resistance were isolated from 44% of the samples of chicken meat that we tested. The statistically significant (P = 0.01) relationship between the presence of phages transducing kanamycin resistance and E. coli isolates resistant to this antibiotic suggests that transduction may be an important mechanism for transferring kanamycin resistance to E. coli. It appears that the transduction of resistance to certain antimicrobials, e.g., kanamycin, not only is widely distributed in E. coli isolates found on meat but also could represent a major mechanism for resistance transfer. The result is of high importance for animal and human health.

Enumeration of total aerobic bacteria and Escherichia coli in minced meat and on carcass surface samples with an automated most-probable-number method compared with colony count protocols

An automated most-probable-number (MPN) system for the enumeration of total bacterial flora and Escherichia coli was compared with plate count agar and tryptone-bile-glucuronide (TBX) and ColiID (in-house method) agar methodology. The MPN partitioning of sample aliquots was done automatically on a disposable card containing 48 wells of 3 different volumes, i.e., 16 replicates per volume. Bacterial growth was detected by the formation of fluorescent 4-methylumbilliferone. After incubation, the number of fluorescent wells was read with a separate device, and the MPN was calculated automatically. A total of 180 naturally contaminated samples were tested (pig and cattle carcass surfaces, n = 63; frozen minced meat, n = 62; and refrigerated minced meat, n = 55). Plate count agar results and MPN were highly correlated (r = 0.99), with log MPN = -0.25 + 1.05 x log CFU (plate count agar) (n = 163; range, 2.2 to 7.5 log CFU/g or cm2). Only a few discrepancies were recorded. In two samples (1.1%), the differences were > or = 1.0 log; in three samples (1.7%), the differences were > or = 0.5 log. For E. coli, regression analysis was done for all three methods for 80 minced meat samples, which were above the limit of detection (1.0 log CFU/g): log MPN = 0.18 + 0.98 x log CFU (TBX), r = 0.96, and log MPN = -0.02 + 0.99 x log CFU (ColiID), r = 0.99 (range, 1.0 to 4.2 log CFU/g). Four discrepant results were recorded, with differences of > 0.5 but < 1.0 log unit. These results suggest that the automated MPN method described is a suitable and labor-saving alternative to colony count techniques for total bacterial flora and E. coli determination in minced meat or on carcass surfaces.

Game meat hygiene in focus: microbiology, epidemiology, risk analysis and quality assurance.

The inspection of animals at slaughter has traditionally been a key element in the production of safe meat for human consumption. Wild boar (Sus scrofa) and red deer (Cervus elaphus), are two farmed game species that undergo meat inspection as governed by European Commission Regulation (EC) no. 854/2004 (EC, 2004). The primary role of meat inspection is to prevent food-borne hazards entering the food chain. Its secondary role is to monitor animal health and welfare. Proposed changes to the European Commission Regulations and their impact on animal health and welfare surveillance were assessed in this work. The diseases and welfare conditions were: foot and mouth disease (FMD), tuberculosis attributed to Mycobacterium bovis (TB), African/classical swine fever (A/CSF) and trauma in wild boar, and FMD, pasteurellosis, yersiniosis, TB, necrobacilliosis, winter death syndrome and trauma/injuries in red deer. Scenario tree analyses were used to calculate the probability of detection for mild and typical cases of the aforementioned diseases, for the current and proposed, visual only, meat inspection protocol. The effectiveness of abattoir surveillance compared with clinical surveillance, for the exotic disease A/CSF in wild boar and endemic disease TB in red deer, was evaluated using scenario trees. The current meat inspection protocol showed a low probability of detecting typical cases of FMD in deer and high detection probabilities for the other aforementioned diseases and welfare conditions. The current protocol was significantly less effective at detecting mild cases rather than typical cases of A/CSF, FMD and TB in wild boar. The visual only protocol significantly decreased the probability of detecting mild and typical cases of TB in deer. Clinical surveillance was more effective than abattoir surveillance at detecting A/CSF, except when high numbers of animals went through the abattoir increasing the overall probability of detection. Meat inspection proved more effective than clinical surveillance at detecting cases of TB in deer.

Carcass and meat quality traits of wild boar (Sus scrofa s. L.) with 2n=36 karyotype compared to those of phenotypically similar crossbreeds (2n=37 and 2n=38) raised under same farming conditions. 1. Carcass quantity and meat dressing

The aim of this study was to compare wild boar (chromosomal number 2n=36) to phenotypically similar animals of 2n=37 and 2n=38 chromosomes (crossbreeds) with respect to live weight, carcass yield, meat yield, fat and weight of inner organs. All animals were born and raised on the same farm and slaughtered at 39 weeks. The final live weight of wild boar 2n=36 was significantly lower (47.2kg) as compared to crossbreeds (80.0kg). Animals 2n=36 had more carcass yields (65.5%) than 2n=37 karyotype (64.9%) and 2n=38 (64.4%). Wild boar had the highest yields for the cuts with bones and boneless cuts compared to crossbreeds. Therefore, variations in karyotype are accompanied by differences in some carcass quantitative traits, i.e., 2n=36 grow and fatten slower than crossbreeds 2n=37 and 2n=38.

Quality traits of wild boar mould-ripened salami manufactured with different selections of meat and fat tissue, and with and without bacterial starter cultures

Quality traits of wild boar mould-ripened salami were assessed in eight batches produced from two different assortments of meat (hind leg vs. shoulder), fat tissue (backfat from domestic pigs vs. fat tissue from wild boar), and with and without addition of a commercial bacterial starter culture. Chemical composition of finished products (day 35) were in compliance with national food codex. Batches produced with a bacterial starter culture were generally preferred by taste panels, had significantly lower concentrations of TBARS (<1.5 mgmalondialdehyde/kg) and peroxide values (POVs) and lower concentrations of cadaverine (<50 mg/kg), histamine (<10 mg/kg) and putrescine (<60 mg/kg). TBARS and POVs were inversely related to sensory preference scores (r(2)=0.84 and 0.88). Batches produced from shoulder muscles contained significantly higher concentrations of cadaverine, histamine and putrescine. TBARS were highest in batches manufactured with fat tissue from wild boars. These findings should be considered when guides to good practice for the manufacture of game meat products are developed.

Biogenic amines and polyamines and total aerobic count during storage of vacuum-packaged porcine kidney, liver and spleen

Biogenic amines and polyamines (cadaverine, histamine, 2-phenylethylamine, putrescine, spermidine, spermine, tryptamine and tyramine) were analysed in vacuum-packaged porcine livers, kidneys and spleens stored at 3°C and 7°C (for up to 6 days) or 0°C (for up to 21 days). Total aerobic count, pH and sensory assessment were done in parallel. While histamine, 2-phenylethylamine and tryptamine concentrations were nearly constant, spermidine and spermine showed a moderately declining trend, irrespective of the storage temperature. Cadaverine, putrescine and tyramine concentrations increased with storage time and temperature. Maximum concentrations at day 21 at 0°C were: 122mg/kg for cadaverine, 207.35mg/kg for putrescine and 63.19mg/kg for tyramine. The correlation of concentrations of the latter three amines and the total aerobic count was ranging from r 0.54 to 0.89. A significant rise in amine concentrations was observed only when the total aerobic count exceeded 6 log10 cfu/g. Concentrations of cadaverine, putrescine and tyramine may be useful to confirm spoilage of vacuum-packaged inner organs. The fraction of the potential food-borne pathogen Aeromonas in high-pH organs (spleen, kidney) during storage was significantly higher than in liver, with low pH. It was observed that the spermine: spermidine ratio of spleen (3:2; weight base) was significantly different from that of liver and kidney (4:1).

The effects of atmospheric pressure cold plasma treatment on microbiological, physical-chemical and sensory characteristics of vacuum packaged beef loin

Effects on vacuum packaged and non-packaged beef longissimus samples exposed to atmospheric cold plasma (ACP) generated at different powers were studied over a 10day period of vacuum-, and a subsequent 3day period of aerobic storage. Exposure of non-covered beef samples under high power ACP conditions resulted in increased a*, b*, Chroma and Hue values, but ACP treatment of packaged loins did not impact colour (L*, a*, b*, Chroma, Hue), lipid peroxidation, sarcoplasmic protein denaturation, nitrate/nitrite uptake, or myoglobin isoform distribution. Colour values measured after 3days of aerobic storage following unpackaging (i.e. 20days post-mortem) were similar and all compliant with consumer acceptability standards. Exposure to ACP of the polyamide-polyethylene packaging film inoculated with Staphylococcus aureus, Listeria monocytogenes and two Escherichia coli strains resulted in >2 log reduction without affecting the integrity of the packaging matrix. Results indicate that ACP can reduce microbial numbers on surfaces of beef packages without affecting characteristics of the packaged beef.

Alaria alata mesocercariae in wild boar (Sus scrofa, Linnaeus, 1758) in south regions of the Czech Republic

From March 2012 to February 2013, 221 wild boar carcasses obtained from regular hunts in the Czech Republic were tested for Alaria alata mesocercariae using the Alaria-migration-technique. Most samples originated from South Moravia (173), and all 15 positive samples were found in this region, in particular in Tvrdonice (3/10) and Lanžhot (12/28), close to the site of the first description of this parasitic stage in wild boars in the territory of former ČSSR. These hunting grounds are located between the rivers March and Thaya, and rich in floodplains. Among the 38 carcasses tested in this area, higher carcass weights were associated with higher frequency of positive carcasses (P<0.05). Overall frequency of positive carcasses was 15/221 (6.8%). In positive samples (adipose and glandular tissue and muscle), the median number of mesocercariae was 14.3 per 100 g (range 3-69).